Beta-nicotinamide mononucleotide liposome as well as preparation method and application thereof

Abstract

The invention relates to a beta-nicotinamide mononucleotide liposome as well as a preparation method and application thereof. The beta-nicotinamide mononucleotide lipidosome comprises a lipidosome carrier and beta-nicotinamide mononucleotide entrapped in the lipidosome carrier, the liposome carrier is prepared from lecithin and hydrogenated lecithin. According to the invention, the liposome carrier is prepared from the composite phospholipid, and the beta-nicotinamide mononucleotide is encapsulated, so that the stability of the beta-nicotinamide mononucleotide liposome is improved. Meanwhile, lecithin and hydrogenated lecithin have a remarkable synergistic effect in the aspect of improving the encapsulation efficiency of beta-nicotinamide mononucleotide and can improve the mechanical strength of the liposome, so that the stability of a beta-nicotinamide mononucleotide liposome product is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanoliposomes, and particularly relates to a beta-nicotinamide mononucleotide liposome as well as a preparation method and application thereof. BACKGROUND

[0002] Beta-nicotinamide mononucleotide (hereinafter referred to as NMN) is a kind of bioactive nucleotide, which is widely present in various organisms, is a direct precursor of NAD + , and can improve the level of NAD + by supplementing NMN. NAD + is associated with human health and aging, is a key coenzyme indispensable in a variety of biochemical reactions in the body, participates in important biological oxidation and energy metabolism processes such as glycolysis, tricarboxylic acid cycle, gluconeogenesis, fatty beta oxidation, ketone body generation and amino acid metabolism, and directly or indirectly affects cell functions such as DNA repair, chromatin remodeling, cell aging and cell immunity. However, NAD + cannot be absorbed by the human body and cannot be supplemented by direct intake, so supplementing exogenous NMN can be an effective alternative, which can effectively improve the concentration of intracellular NAD + . However, the stability of NMN is easily affected by environmental conditions, and is prone to degradation in aqueous solution due to oxidation, hydrolysis or photolysis, making it difficult to ensure the effectiveness and safety of the product. Therefore, it is necessary to develop a stable liposome to improve the bioavailability of NMN.

[0003] CN117257679A discloses an anti-aging whitening liposome, which comprises a lipid wrapping layer and an efficacy component in the lipid wrapping layer, the efficacy component comprising yeast / rice fermentation product filtrate, golden microalgae and beta-nicotinamide mononucleotide, and the raw material of the lipid wrapping layer comprises lecithin. However, the liposome selects a single lecithin as the lipid wrapping layer, the mechanical strength of the wrapping layer is insufficient, the encapsulation efficiency is low, and the stability of the liposome is poor.

[0004] Therefore, how to develop a beta-nicotinamide mononucleotide liposome to improve the encapsulation efficiency of beta-nicotinamide mononucleotide and increase the stability of the beta-nicotinamide mononucleotide liposome has become one of the technical problems to be solved at present. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a beta-nicotinamide mononucleotide liposome as well as a preparation method and application thereof. The beta-nicotinamide mononucleotide liposome is wrapped with beta-nicotinamide mononucleotide, which increases the stability of the beta-nicotinamide mononucleotide liposome, thereby promoting the beta-nicotinamide mononucleotide to exert its anti-aging effect and the like.

[0006] To achieve the object of the present application, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a beta-nicotinamide mononucleotide liposome, which comprises a liposome carrier and a beta-nicotinamide mononucleotide encapsulated in the liposome carrier.

[0008] The raw materials for preparing the liposome carrier include lecithin and hydrogenated lecithin.

[0009] The lecithin includes any one of egg yolk lecithin, soybean lecithin or oat lecithin, or a combination of at least two thereof.

[0010] The lecithin is derived from egg yolk, soybean or oat, has good hydrophilicity and lipophilicity, can form a bilayer structure in the cell membrane, and plays a role in maintaining the morphology and function of the cell; the hydrogenated lecithin is a product prepared by hydrogenation of lecithin, which improves the hydrophilicity of phospholipids and has stronger emulsifying effect.

[0011] The present application creatively finds that lecithin and hydrogenated lecithin have a significant synergistic effect in improving the encapsulation rate of NMN, and at the same time, by preparing the liposome carrier with complex phospholipids, the mechanical strength of the liposome can be improved, thereby improving the stability of the beta-nicotinamide mononucleotide liposome product.

[0012] Preferably, the mass ratio of the lecithin to the hydrogenated lecithin is (4-12):(0.5-4).

[0013] Specific point values in 4-12 can be selected as 4, 5, 6, 7, 8, 9, 10, 11, 12, etc., and specific point values in 0.5-4 can be selected as 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.

[0014] Preferably, the lecithin includes a combination of egg yolk lecithin and soybean lecithin, and the mass ratio of the egg yolk lecithin to the soybean lecithin is (1-5):(3-7).

[0015] Specific point values in 1-5 can be selected as 1, 2, 3, 4, 5, etc., and specific point values in 3-7 can be selected as 3, 4, 5, 6, 7, etc.

[0016] The present application creatively finds that the combination of egg yolk lecithin and soybean lecithin and the hydrogenated lecithin can better exert the synergistic effect of lecithin and hydrogenated lecithin in improving the encapsulation effect of NMN. In addition, when the egg yolk lecithin and the soybean lecithin are compounded according to the above ratio, the encapsulation rate of NMN can be better improved.

[0017] Preferably, the raw materials for preparing the liposome carrier further include a stabilizer.

[0018] Preferably, the stabilizer comprises any one or a combination of at least two of cholesterol, Tween, or vitamin E.

[0019] In the preparation of β-nicotinamide mononucleotide liposomes (hereinafter referred to as NMN liposomes), a stabilizer is added to enhance the compactness of the liposome membrane through hydrogen bonding and hydrophobic interactions, thereby inhibiting NMN leakage. At the same time, it can enhance the stability of the bilayer structure of the liposome membrane and improve the stability of the liposome carrier and the permeability of NMN.

[0020] Preferably, the stabilizer comprises a combination of cholesterol, Tween, and vitamin E.

[0021] This invention creatively discovers that cholesterol, Tween, and vitamin E have a significant synergistic effect in enhancing the stability of the liposome membrane bilayer structure. The three together act as stabilizers, which can further improve the stability of the liposome carrier and the permeability of NMN.

[0022] Preferably, the Tween includes Tween-80.

[0023] Preferably, the mass ratio of cholesterol, Tween, and vitamin E is (1-3):(0.1-4):(0.5-1).

[0024] Among them, the specific point values ​​in 1-3 can be 1, 1.5, 2, 2.5, 3, etc.; the specific point values ​​in 0.1-4 can be 0.1, 1, 2, 3, 4, etc.; and the specific point values ​​in 0.5-1 can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.

[0025] Preferably, the mass ratio of phospholipid to stabilizer in the liposome carrier is 15:(1-10).

[0026] The specific point values ​​from 1 to 10 can be selected as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0027] In a second aspect, the present invention provides a method for preparing β-nicotinamide mononucleotide liposomes as described in the first aspect, the method comprising:

[0028] The liposome carrier and solvent were mixed and rotary evaporated to obtain a lipid membrane; the lipid membrane was then hydrated with a solution containing NMN to obtain NMN liposomes.

[0029] Preferably, the solvent includes anhydrous ethanol.

[0030] This invention uses ethanol as a solvent to dissolve liposome carriers, which has low toxicity, is easily volatile, and poses no risk of residual toxicity, thus improving the safety of product application.

[0031] Preferably, the mass-to-volume ratio of the liposome carrier to the solvent is (1-5):(10-50) g / mL.

[0032] Among them, the specific point values ​​in 1-5 can be 1, 2, 3, 4, 5, etc., and the specific point values ​​in 10-50 can be 10, 20, 30, 40, 50, etc.

[0033] Preferably, the solution containing NMN comprises an aqueous solution of NMN.

[0034] Preferably, the NMN content in the solution containing NMN is 30-70% by mass, for example, it can be 30%, 40%, 50%, 60%, 70%, etc.

[0035] Preferably, the hydration treatment temperature is 35-45℃ and the hydration treatment time is 15-25 min.

[0036] Among them, the specific point values ​​in the 35-45℃ range can be 35℃, 37℃, 39℃, 41℃, 43℃, 45℃, etc., and the specific point values ​​in the 15-25min range can be 15 min, 17 min, 19 min, 21 min, 23 min, 25 min, etc.

[0037] This invention, through precise control of hydration treatment parameters, can further enhance the encapsulation effect of liposomes on NMN, and promote the anti-aging effects of NMN liposomes.

[0038] Preferably, the hydration treatment further includes homogenization and membrane treatment.

[0039] Preferably, the pressure of the homogenization process is 30-70 MPa, for example, it can be 30 MPa, 40 MPa, 50 MPa, 60 MPa, 70 MPa, etc.

[0040] Preferably, the pore size of the membrane treatment is 100-450 nm, for example, it can be 100 nm, 220 nm, 450 nm, etc.

[0041] Thirdly, the present invention provides a β-nicotinamide mononucleotide liposome lyophilized powder, wherein the raw materials for preparing the β-nicotinamide mononucleotide liposome lyophilized powder include the β-nicotinamide mononucleotide liposomes and lyophilization protectant described in the first aspect.

[0042] In both the first and second aspects of this invention, stable encapsulation of the active ingredient NMN is achieved through liposome encapsulation technology. Because NMN is relatively easy to deactivate, it is further prepared into a lyophilized powder for preservation. The NMN liposomes described in the first aspect of this invention, or the NMN liposomes prepared by the method described in the second aspect, improve the stability of NMN through the synergistic effect of the components in the formulation, while solving the problem of lyophilization collapse during the preparation of liposome lyophilized powder.

[0043] Preferably, the freeze-drying protectant includes trehalose and mannitol.

[0044] Trehalose is a non-reducing disaccharide with antioxidant and moisturizing properties, as well as a strong hydration capacity, which can improve the stability of freeze-drying systems. Mannitol is a six-carbon polyol with moisturizing and skin-repairing effects, and can also be used as a freeze-drying excipient, providing excellent support.

[0045] On the one hand, the present invention selects trehalose and mannitol as freeze-drying protectants. The two have a significant synergistic effect in improving the stability of β-nicotinamide mononucleotide liposome freeze-dried powder and can maintain the activity of NMN for a long time.

[0046] On the other hand, this invention creatively discovers that trehalose, mannitol and NMN have a significant synergistic effect in anti-aging. The two can not only improve the stability of NMN liposomes, but also work synergistically with NMN to exert anti-aging effects.

[0047] Preferably, the mass ratio of trehalose to mannitol is (1-10):(1-5).

[0048] Preferably, the mass ratio of the NMN liposomes to the lyophilization protectant is 100:(6-10).

[0049] Among them, the specific point values ​​in 1-10 can be 1, 3, 5, 7, 9, 10, etc., the specific point values ​​in 1-5 can be 1, 2, 3, 4, 5, etc., and the specific point values ​​in 6-10 can be 6, 7, 8, 9, 10, etc.

[0050] Fourthly, the present invention provides a method for preparing β-nicotinamide mononucleotide liposome lyophilized powder as described in the third aspect, the preparation method comprising:

[0051] The NMN liposomes described in the first aspect are mixed with a freeze-drying protectant and then freeze-dried to obtain the NMN liposome freeze-dried powder.

[0052] Fifthly, the present invention provides the application of β-nicotinamide mononucleotide liposomes as described in the first aspect or β-nicotinamide mononucleotide liposome lyophilized powder as described in the third aspect in cosmetics, food or health products.

[0053] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] This invention utilizes a composite phospholipid to prepare a liposome carrier for encapsulating NMN, thereby increasing the stability of the NMN liposomes. Simultaneously, lecithin and hydrogenated lecithin exhibit a significant synergistic effect in improving the encapsulation rate of NMN and can also enhance the mechanical strength of the liposomes, thus improving the stability of the NMN liposome product.

[0056] Furthermore, adding stabilizers to the liposome carrier enhances the compactness of the liposome membrane through hydrogen bonding and hydrophobic interactions, inhibiting NMN leakage. Simultaneously, it enhances the stability of the liposome membrane bilayer structure, improving both the stability of the liposome carrier and the permeability of NMN. In addition, cholesterol, Tween, and vitamin E have significant synergistic effects in enhancing the stability of the liposome membrane bilayer structure; together, these three act as stabilizers, further improving the stability of the liposome carrier and the permeability of NMN.

[0057] Furthermore, in the preparation process of NMN liposomes, precise control of hydration parameters can further enhance the encapsulation effect of liposomes on NMN, promoting the anti-aging effects of NMN liposomes.

[0058] Furthermore, to ensure the activity of NMN, this invention further prepares NMN liposomes into freeze-dried powder for preservation. This invention selects trehalose and mannitol as freeze-drying protectants. The two have a significant synergistic effect in improving the stability of NMN liposome freeze-dried powder and can maintain the activity of NMN for a long time. At the same time, trehalose, mannitol and NMN have a significant synergistic effect in anti-aging. The two can not only improve the stability of NMN liposomes, but also work together with NMN to exert anti-aging effects. Attached Figure Description

[0059] Figure 1 These are transmission electron microscopy (TEM) images of the NMN liposome lyophilized powder tested on day 0 and day 30 of Test Example 2. Detailed Implementation

[0060] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0061] The sources of each component / raw material in the following embodiments are shown in Table 1.

[0062] Table 1

[0063]

[0064] Preparation Example 1

[0065] This preparation example provides a β-nicotinamide mononucleotide liposome, prepared by the following method:

[0066] 10 g of phospholipids (egg yolk lecithin, soybean lecithin, and hydrogenated lecithin in a mass ratio of 3:5:2) and 5 g of stabilizers (cholesterol, Tween-80, and vitamin E in a mass ratio of 2:1:0.8) were dissolved in 100 mL of anhydrous ethanol and evaporated at 50 °C (50 rpm, 0.1 MPa) until a film was formed, thus obtaining a lipid membrane. 50 mL of a 50% NMN aqueous solution was added and hydrated at 40 °C for 20 min. The mixture was then subjected to ice bath sonication at 300 W for 20 min (10 seconds on / 10 seconds off), homogenized using a high-pressure homogenizer (50 MPa, 4 cycles), and passed through a 220 nm membrane to obtain β-nicotinamide mononucleotide liposomes (hereinafter referred to as NMN liposomes).

[0067] Preparation Example 2

[0068] This preparation example provides an NMN liposome, prepared by the following method:

[0069] 10 g of phospholipids (egg yolk lecithin, soybean lecithin, and hydrogenated lecithin in a mass ratio of 1:3:4) and 6.5 g of stabilizers (cholesterol, Tween-80, and vitamin E in a mass ratio of 3:4:1) were dissolved in 200 mL of anhydrous ethanol and evaporated at 50 °C (50 rpm, 0.1 MPa) until a film was formed, thus obtaining a lipid membrane. 50 mL of a 70% NMN aqueous solution was added and hydrated at 45 °C for 15 min. The mixture was then subjected to ice bath sonication at 400 W for 10 min (10 seconds on / 10 seconds off), homogenized using a high-pressure homogenizer (70 MPa, 3 cycles), and passed through a 450 nm membrane to obtain NMN liposomes.

[0070] Preparation Example 3

[0071] This preparation example provides an NMN liposome, prepared by the following method:

[0072] 10 g of phospholipids (egg yolk lecithin, soybean lecithin, and hydrogenated lecithin in a mass ratio of 5:7:0.5) and 0.7 g of stabilizers (cholesterol, Tween-80, and vitamin E in a mass ratio of 1:1:0.5) were dissolved in 80 mL of anhydrous ethanol and evaporated at 55 °C (45 rpm, 0.1 MPa) until a film was formed, thus obtaining a lipid membrane. 50 mL of a 40% NMN aqueous solution was added and hydrated at 35 °C for 25 min. The mixture was then subjected to ice bath sonication at 200 W for 30 min (10 seconds on / 10 seconds off), homogenized using a high-pressure homogenizer (30 MPa, 5 cycles), and passed through a 220 nm membrane to obtain NMN liposomes.

[0073] Preparation Example 4

[0074] This preparation example provides an NMN liposome, which differs from Preparation Example 1 only in that the total amount of phospholipids remains unchanged, and soybean lecithin is replaced with an equal amount of egg yolk lecithin. That is, the phospholipid composition is egg yolk lecithin and hydrogenated lecithin in a mass ratio of 8:2. The rest of the preparation method is the same as that of Preparation Example 1.

[0075] Preparation Example 5

[0076] This preparation example provides an NMN liposome, which differs from Preparation Example 1 only in that the total amount of phospholipids remains unchanged, and egg yolk lecithin is replaced with an equal amount of soybean lecithin. That is, the phospholipid composition is soybean lecithin and hydrogenated lecithin in a mass ratio of 8:2. The rest of the preparation method is the same as that of Preparation Example 1.

[0077] Preparation Example 6

[0078] This preparation example provides an NMN liposome, which differs from Preparation Example 1 only in that the total mass of the stabilizer remains unchanged, and the stabilizer is replaced with cholesterol and Tween-80 in a mass ratio of 2:1. The rest of the preparation method is the same as that of Preparation Example 1.

[0079] Preparation Example 7

[0080] This preparation example provides an NMN liposome, which differs from Preparation Example 1 only in that the total mass of the stabilizer remains unchanged, and the stabilizer is replaced with cholesterol and vitamin E in a mass ratio of 2:0.8. The rest of the preparation method is the same as that of Preparation Example 1.

[0081] Preparation Example 8

[0082] This preparation example provides an NMN liposome, which differs from Preparation Example 1 only in that the total mass of the stabilizer remains unchanged, and the stabilizer is replaced with Tween-80 and vitamin E in a mass ratio of 1:0.8. The rest of the preparation method is the same as that of Preparation Example 1.

[0083] Preparation Example 9

[0084] This preparation example provides an NMN liposome, which differs from Preparation Example 1 only in that no stabilizer is added and the phospholipid is directly dissolved in anhydrous ethanol. The rest of the preparation method is the same as that of Preparation Example 1.

[0085] Preparation Example 10

[0086] This preparation example provides an NMN liposome, which differs from Preparation Example 1 only in that the hydration temperature is adjusted to 30°C, while the remaining steps are the same as in Preparation Example 1.

[0087] Preparation Example 11

[0088] This preparation example provides an NMN liposome, which differs from Preparation Example 1 only in that the hydration temperature is adjusted to 50°C, while the remaining steps are the same as in Preparation Example 1.

[0089] Comparative Preparation Example 1

[0090] This preparation example provides an NMN liposome, which differs from Preparation Example 1 only in that the total mass of the liposome carrier phospholipid remains unchanged, and the phospholipid is replaced with egg yolk lecithin and soybean lecithin in a mass ratio of 3:5. The rest of the preparation method is the same as that of Preparation Example 1.

[0091] Comparative Preparation Example 2

[0092] This preparation example provides an NMN liposome, which differs from Preparation Example 1 only in that the total mass of the liposome carrier phospholipid remains unchanged, and the phospholipid is replaced with hydrogenated lecithin. The rest of the preparation method is the same as that of Preparation Example 1.

[0093] Example 1

[0094] This embodiment provides a β-nicotinamide mononucleotide liposome lyophilized powder, prepared by the following method:

[0095] The NMN liposomes obtained in Preparation Example 1 were mixed with 8% of the NMN liposomes by weight of a freeze-drying protectant (trehalose and mannitol in a mass ratio of 5:3), pre-frozen at -50°C for 3 h, then dried at 25°C for 1 Pa for 16 h, and finally desorbed at 45°C for 0.5 Pa for 6 h to obtain β-nicotinamide mononucleotide liposome freeze-dried powder (hereinafter referred to as NMN liposome freeze-dried powder).

[0096] Example 2

[0097] This embodiment provides an NMN liposome lyophilized powder, prepared by the following method:

[0098] The NMN liposomes obtained in Preparation Example 2 were mixed with 10% of the mass of the NMN liposomes in a freeze-drying protectant (trehalose and mannitol in a mass ratio of 2:5), pre-frozen at -50°C for 3 h, then dried at 25°C for 1 Pa for 16 h, and finally desorbed at 45°C for 0.5 Pa for 6 h to obtain NMN liposome freeze-dried powder.

[0099] Example 3

[0100] This embodiment provides an NMN liposome lyophilized powder, prepared by the following method:

[0101] The NMN liposomes obtained in Preparation Example 3 were mixed with 6% by weight of a freeze-drying protectant (trehalose and mannitol in a 1:1 mass ratio) and pre-frozen at -50°C for 3 h, then dried at 25°C for 1 Pa for 16 h, and finally desorbed at 45°C for 0.5 Pa for 6 h to obtain NMN liposome freeze-dried powder.

[0102] Example 4-11

[0103] Examples 4-11 each provide a freeze-dried NMN liposome powder, the only difference from Example 1 being that the NMN liposomes obtained in Example 1 are replaced with an equal mass of the NMN liposomes obtained in Examples 4-11.

[0104] Example 12

[0105] This embodiment provides an NMN liposome lyophilized powder, which differs from Example 1 only in that mannitol is removed from the lyophilization protectant, that is, the total mass of the lyophilization protectant remains unchanged, and the lyophilization protectant is replaced with a single trehalose.

[0106] Example 13

[0107] This embodiment provides an NMN liposome lyophilized powder, which differs from Example 1 only in that trehalose is removed from the lyophilization protectant, that is, the total mass of the lyophilization protectant remains unchanged, and the lyophilization protectant is replaced with a single mannitol.

[0108] Comparative Examples 1-2

[0109] Comparative Examples 1 and 2 each provide a freeze-dried NMN liposome powder. The only difference between them and Example 1 is that the NMN liposomes obtained in Example 1 are replaced with an equal mass of the NMN liposomes obtained in Comparative Examples 1 and 2.

[0110] Comparative Example 3

[0111] This comparative example provides a freeze-dried NMN liposome powder, which differs from Example 1 only in that no freeze-drying protectant is added, and the NMN liposomes obtained in Example 1 are directly freeze-dried. The freeze-drying parameters are consistent with those in Example 1.

[0112] Application Example 1

[0113] This application example provides a skincare essence, formulated by weight percentage as follows: 5% NMN liposome lyophilized powder obtained in Example 1, 3% sodium hyaluronate, 5% panthenol, 3% propylene glycol, 10% glycerin, and the balance being water. The components are homogenized to obtain the skincare essence.

[0114] Application Example 2-13

[0115] Application Examples 2-13 each provide a skin care essence, the only difference from Application Example 1 being that the NMN liposome lyophilized powder prepared in Example 1 is replaced with an equal mass of NMN liposome lyophilized powder prepared in Examples 2-13, while the rest of the formula remains the same as Application Example 1.

[0116] Comparative Application Examples 1-3

[0117] Comparative Application Examples 1-3 each provide a skin care essence, the only difference between them and Application Example 1 is that the NMN liposome lyophilized powder prepared in Example 1 is replaced with an equal mass of NMN liposome lyophilized powder prepared in Comparative Examples 1-3 respectively, while the rest of the formula is the same as Application Example 1.

[0118] Comparative Application Example 4

[0119] Comparative Application Example 4 provides a skin care essence, which differs from Application Example 1 only in that 5% of the NMN liposome freeze-dried powder prepared in Example 1 is replaced with 3.1% trehalose and 1.9% mannitol, while the rest of the formula remains the same as Application Example 1.

[0120] Test Example 1

[0121] Encapsulation efficiency test:

[0122] (1) Preparation of NMN standard curve: Take NMN sample and prepare standard solutions with deionized water with concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 5 mg / mL respectively. Then use a spectrophotometer to test the absorbance of the standard solution at 287 nm at 25 °C. Plot the standard curve with ultraviolet absorbance as Y axis and NMN concentration (mg / mL) as X axis.

[0123] (2) Determination of free NMN content: The NMN liposomes prepared in Preparation Examples 1-11 and Comparative Preparation Examples 1-2 were placed in ultrafiltration centrifuge tubes and centrifuged at low temperature and high speed (4℃, 20000 rpm for 45 min). The absorbance of the filtrate was measured at a wavelength of 287 nm, and the free NMN content was calculated by combining the standard curve.

[0124] (3) Encapsulation rate calculation: The amount of NMN added during the preparation of liposomes is taken as the total amount of NMN in the liposomes, and the encapsulation rate is calculated according to the following formula.

[0125]

[0126] The encapsulation efficiency test results of NMN liposomes obtained from each preparation example and the comparative preparation example are shown in Table 2.

[0127] Table 2

[0128]

[0129] The data from Preparation Examples 1, 4-5 and Comparative Preparation Examples 1-2 in Table 2 show that the combination of lecithin and hydrogenated lecithin, especially the combination of egg yolk lecithin, soybean lecithin and hydrogenated lecithin to prepare liposome carriers, has a significant synergistic effect in improving the encapsulation effect of NMN.

[0130] A comparison of the data from Preparation Example 1 with those from Preparation Examples 6-9 shows that the addition of stabilizers during the preparation of NMN liposomes enhances the density of the phospholipid membrane of the liposome carrier, thereby improving the encapsulation efficiency of NMN. Simultaneously, cholesterol, Tween-80, and vitamin E are added to the liposomes as stabilizers, and these three components exhibit a significant synergistic effect in improving the NMN encapsulation efficiency.

[0131] A comparison of the data from Preparation Example 1 and Preparation Examples 10-11 shows that controlling the hydration temperature at 35-45℃ during the preparation of NMN liposomes can further enhance the encapsulation effect of liposomes on NMN.

[0132] Test Example 2

[0133] Stability test:

[0134] (1) Evaluation of particle size stability:

[0135] The NMN liposome lyophilized powder prepared in Example 1 was stored at 10°C for 30 days and then dissolved in deionized water to obtain an aqueous solution with a mass fraction of 0.2%.

[0136] (1.1) The particle size was analyzed before testing (day 0) and after 30 days of storage (day 30) using a dynamic light scattering analyzer (DLS). The particle size was sonicated for 20 min before each particle size analysis.

[0137] The particle size test results on day 0 and day 30 are shown in Table 3. It can be seen that the NMN liposome freeze-dried powder product involved in this invention has little particle size change after being placed at 10°C for 30 days, and has good particle size uniformity (PDI<0.15), showing excellent stability.

[0138] Table 3

[0139]

[0140] (1.2) The NMN liposome lyophilized powder samples before testing (day 0) and after 30 days of storage (day 30) were characterized using transmission electron microscopy. The results are as follows: Figure 1 As shown, the NMN liposome lyophilized powder product involved in this invention is spherical under transmission electron microscopy, with uniform distribution, clear boundaries, and a particle size of (100±2) nm, which is consistent with the particle size detection results of DLS. According to the transmission electron microscopy results, the NMN liposomes did not undergo significant morphological changes after 30 days of storage, exhibiting excellent stability characteristics.

[0141] (2) NMN residual rate test:

[0142] The NMN liposome lyophilized powders prepared in Examples 1-13 and Comparative Examples 1-2 were subjected to accelerated testing (40℃ / 75% RH) for 30 days at 40℃ and 75% RH. After the 30-day test, the residual rate of NMN in the lyophilized powder samples was measured.

[0143] (2.1) Preparation of NMN standard curve: Take NMN sample and prepare standard solutions with deionized water with concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 5 mg / mL respectively. Then use a spectrophotometer to test the absorbance of the standard solution at 25℃ and 287nm. With the ultraviolet absorbance intensity as the Y axis and the NMN concentration (mg / mL) as the X axis, prepare the standard curve.

[0144] (2.2) Determination of residual NMN content: Take the lyophilized liposome powder sample, add methanol to it to prepare a 4 mg / mL solution, and then place it in an ultrafiltration centrifuge tube. Centrifuge at low temperature and high speed (4℃, 20000 rpm for 45 min). Take the filtrate and detect the absorbance at a wavelength of 287 nm. Calculate the NMN content by combining it with the standard curve. This is the residual NMN content.

[0145] (2.3) Calculation of residual rate: The amount of NMN added during the preparation of liposomes is taken as the total amount of NMN in liposomes, and the residual rate is calculated according to the following formula.

[0146]

[0147] The test results are shown in Table 4.

[0148] Table 4

[0149]

[0150] As can be seen from the comparison of the data of Examples 1, 4-5 and Comparative Examples 1-2 in Table 4, lecithin and hydrogenated lecithin work together, especially egg yolk lecithin, soybean lecithin and hydrogenated lecithin are used together to prepare liposome carriers, which has a significant synergistic effect in improving the stability of NMN.

[0151] A comparison of the data from Examples 1 and 6-9 shows that the addition of stabilizers during the preparation of NMN liposomes can further enhance the compactness of the phospholipid membrane of the liposome carrier and the long-term storage stability of the liposomes. Simultaneously, cholesterol, Tween-80, and vitamin E are added to the liposomes as stabilizers, and the three have a significant synergistic effect in improving the stability of NMN liposomes.

[0152] A comparison of the data from Example 1 and Examples 10-11 shows that controlling the hydration temperature at 35-45℃ during the preparation of NMN liposomes can further improve the stability of the prepared lyophilized liposome powder.

[0153] A comparison of the data from Examples 1, 12-13, and Comparative Example 3 shows that adding a freeze-drying protectant during the preparation of NMN liposome freeze-dried powder can further improve the stability of the NMN liposome freeze-dried powder. Simultaneously, mannitol and trehalose, both added as freeze-drying protectants, exhibit a significant synergistic effect in improving the stability of the NMN liposome freeze-dried powder.

[0154] Test Example 3

[0155] Security testing:

[0156] One hundred and seventy subjects aged 20-45 years were randomly selected, including 74 males and 96 females. Using a closed patch test, 0.025 g of the serum products from Application Examples 1-13 and Control Application Examples 1-4 were placed in a patch applicator (with deionized water as the control). The patch was applied to the flexor side of the subject's forearm with hypoallergenic adhesive tape. The test products were removed after 24 hours. Skin reactions were observed at 0.5, 24, and 48 hours after removal, and the results were recorded according to the skin reaction grading standards in the *Cosmetic Safety Technical Specifications* (2015 edition). The results are shown in Table 5.

[0157] Table 5

[0158]

[0159] The above results indicate that the skin of all subjects in different groups showed negative reactions, confirming that the NMN liposome lyophilized powder involved in this invention is safe, mild, and non-irritating to the skin.

[0160] Test Example 4

[0161] Anti-aging effect test:

[0162] In the randomized test example 3, 170 subjects with noticeable fine lines and wrinkles on their faces, accompanied by sagging, were divided into 17 groups. After cleansing their skin, each group applied an appropriate amount of the serum product from Examples 1-13 and the control examples 1-4 evenly around the eyes, gently massaging until fully absorbed. This was done twice daily, morning and evening, for four consecutive weeks. Skin images were captured using the VISIA-CR facial image analyzer to assess changes in the area and depth of wrinkles in the crow's feet region. The test results are shown in Table 6.

[0163] Table 6

[0164]

[0165] As shown in Table 6, when the NMN liposome freeze-dried powder involved in this invention is applied to skin care products, the resulting products have significant anti-aging effects.

[0166] A comparison of the data from Application Example 1 with comparative Application Examples 3 and 4 shows that the NMN liposomes involved in this invention have a significant synergistic effect with mannitol and trehalose in anti-aging. A comparison of the data from Application Example 1 with Application Examples 12-13 shows that mannitol and trehalose, acting together as freeze-drying protectants, improve the stability of NMN liposomes and enhance their synergistic anti-aging effect. These results indicate that freeze-drying protectants not only improve the stability of liposomes but also synergistically enhance anti-aging effects with NMN.

[0167] The data from Application Example 1, Application Example 4-5 and Comparative Application Example 1-2 show that the liposome carrier prepared by combining lecithin and hydrogenated lecithin, especially egg yolk lecithin, soybean lecithin and hydrogenated lecithin, can better maintain the activity of NMN and has a significant synergistic effect in improving anti-aging effects.

[0168] A comparison of the data from Application Example 1 and Application Examples 6-9 shows that the addition of stabilizers can improve the encapsulation efficiency of NMN in the liposome carrier during the preparation of NMN liposomes. Simultaneously, cholesterol, Tween-80, and vitamin E are added to the liposomes as stabilizers, and these three have a significant synergistic effect in improving the NMN encapsulation efficiency, thereby enhancing the anti-aging effect of the NMN liposomes.

[0169] A comparison of the data from Application Example 1 and Application Examples 10-11 shows that controlling the hydration temperature at 35-45℃ during the preparation of NMN liposomes can further slow down the degradation of NMN, thereby improving the protective and anti-aging effects of NMN liposomes.

[0170] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0171] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0172] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A β-nicotinamide mononucleotide liposome, characterized in that, The β-nicotinamide mononucleotide liposome includes a liposome carrier and β-nicotinamide mononucleotide encapsulated in the liposome carrier; The raw materials for preparing the liposome carrier include lecithin and hydrogenated lecithin; The lecithin includes any one or a combination of at least two of egg yolk lecithin, soy lecithin, or oat lecithin.

2. The β-nicotinamide mononucleotide liposome according to claim 1, characterized in that, The mass ratio of the lecithin to hydrogenated lecithin is (4-12):(0.5-4); Preferably, the lecithin comprises a combination of egg yolk lecithin and soybean lecithin; Preferably, the mass ratio of egg yolk lecithin to soybean lecithin is (1-5):(3-7).

3. The β-nicotinamide mononucleotide liposome according to claim 1 or 2, characterized in that, The raw materials for preparing the liposome carrier also include stabilizers; Preferably, the stabilizer comprises any one or a combination of at least two of cholesterol, Tween, or vitamin E; Preferably, the stabilizer comprises a combination of cholesterol, Tween, and vitamin E; Preferably, the mass ratio of cholesterol, Tween, and vitamin E is (1-3):(0.1-4):(0.5-1); Preferably, the mass ratio of phospholipid to stabilizer in the liposome carrier is 15:(1-10).

4. A method for preparing β-nicotinamide mononucleotide liposomes as described in any one of claims 1-3, characterized in that, The preparation method includes: The liposome carrier and solvent were mixed and rotary evaporated to obtain a lipid membrane; the lipid membrane was then hydrated with a solution containing β-nicotinamide mononucleotide to obtain β-nicotinamide mononucleotide liposomes.

5. The preparation method according to claim 4, characterized in that, The solvent includes anhydrous ethanol; Preferably, the mass-to-volume ratio of the liposome carrier to the solvent is (1-5):(10-50) g / mL; Preferably, the solution containing β-nicotinamide mononucleotide comprises an aqueous solution of β-nicotinamide mononucleotide; Preferably, the solution containing β-nicotinamide mononucleotide has a β-nicotinamide mononucleotide content of 30-70% by mass.

6. The preparation method according to claim 4 or 5, characterized in that, The hydration treatment temperature is 35-45℃, and the hydration treatment time is 15-25 min.

7. The preparation method according to any one of claims 4-6, characterized in that, The hydration treatment also includes homogenization and membrane treatment; Preferably, the pressure of the homogenization process is 30-70 MPa; Preferably, the pore size of the membrane treatment is 100-450 nm.

8. A β-nicotinamide mononucleotide liposome lyophilized powder, characterized in that, The raw materials for preparing the β-nicotinamide mononucleotide liposome lyophilized powder include β-nicotinamide mononucleotide liposomes and lyophilization protectant as described in any one of claims 1-3; Preferably, the freeze-drying protectant includes trehalose and mannitol; Preferably, the mass ratio of trehalose to mannitol is (1-10):(1-5); Preferably, the mass ratio of the β-nicotinamide mononucleotide liposome to the lyophilization protectant is 100:(6-10).

9. A method for preparing the β-nicotinamide mononucleotide liposome lyophilized powder as described in claim 8, characterized in that, The preparation method includes: Mix the β-nicotinamide mononucleotide liposomes according to any one of claims 1-3 with a lyophilization protectant and freeze-dry to obtain the β-nicotinamide mononucleotide liposome lyophilized powder.

10. The use of a β-nicotinamide mononucleotide liposome as described in any one of claims 1-3 or the β-nicotinamide mononucleotide liposome lyophilized powder as described in claim 8 in cosmetics, food or health products.

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